PROJECT SUMMARY/ABSTRACT ! Proteomics is critical for health research as it enables analysis of proteins involved in a broad range of biological processes. Although proteomics has laid the foundation for many advances in disease diagnosis, treatment, and prevention, improvements in sample preparation and processing of protein and sugar biomolecules have lagged far behind impressive developments in mass spectrometry (MS) technologies. Most proteomics laboratories are heavily reliant on enzyme technologies that are over 75 years old, namely trypsin. While trypsin has been a very powerful molecular tool, the potential of new classes and capabilities for relevant enzymes have yet to be explored and realized. At CinderBio, we have developed technologies that yield enzymes with remarkable stability and activity at high temperatures (70-105C) and extremes in acidic pH (1.5 - 5) that also tolerate an unprecedented spectrum of chemicals and solvents. These newly available enzymes hold great promise to rapidly advance many areas of proteomics research. Importantly, optimal conditions for our enzymatic activities are conditions that denature and solubilize most if not all mesophilic proteins, a current limitation in sample processing for some MS analyses. At CinderBio, we produce a variety of glycohydrolases, including sugar-de-branching enzymes, proteases, and other classes of hyper-stable enzymes that; 1) function optimally at extreme temperatures and pH that assist in exposing target bonds (i.e. conditions where mesophilic proteins are denatured), 2) minimally cross-react and have little autolysis, allowing stable multi-enzyme formulations, and 3) are tolerant of many harsh oxidizing/reducing chemicals, detergents, and solvents, and 4) have very fast reaction rates. These capabilities open possibilities for entirely new methods for proteomic sample preparation for mass spectrometry. CinderBio enzyme formulations promise to reduce required sample sizes, increase coverage, make quantitation more reliable, simultaneously de-glycosylate and proteolyze samples, and generally streamline upstream sample preparation. Here we propose to formulate our enzymes for the explicit purpose of modernizing the proteomic methodologies of sample preparation. We see immediate opportunities to address the growing list of difficult target proteins like histones, transmembrane and glycosylated proteins (e.g. neural tissues) while increasing digestion efficiencies. These Phase I experiments will focus on establishing proteomics protocols for our enzymes on a set of well-known substrates, investigating simultaneous deglycosylation and proteolysis of a set of glycoproteins, and benchmarking for performance against standard tryptic protocols and enzymes.